Aug 4, 1992 - Salmonella typhimurium. BARRY S. GOLDMANt* AND JOHN R. ROTH. Department ofBiology, University of Utah, Salt Lake City, Utah 84112.
JOURNAL OF BACrERIOLOGY, Mar. 1993, p. 1457-1466
Vol. 175, No. 5
0021-9193/93/051457-10$02.00/0 Copyright C) 1993, American Society for Microbiology
Genetic Structure and Regulation of the cysG Gene in Salmonella typhimurium BARRY S. GOLDMANt* AND JOHN R. ROTH Department of Biology, University of Utah, Salt Lake City, Utah 84112 Received 4 August 1992/Accepted 27 December 1992
Siroheme, a cofactor of both sulfite and nitrite reductase in Sabnonella typhimurium, requires the cysG gene for its synthesis. Three steps are required to synthesize siroheme from uroporphyrinogen m, the last common intermediate in the heme and siroheme pathways. All previously characterized cysG mutants were shown to be defective for the synthesis of cobalamin (B12), which shares a common precursor with siroheme. Since few cysG auxotrophs had been previously analyzed and since there is no evidence of siroheme mutants outside of the cysG region, we sought to expand the analysis of the region by isolating more mutations and studying the transcriptional regulation of the cysG gene using IacZ fusions. We isolated and analyzed 66 cysG auxotrophs. All were defective for both siroheme and cobalamin synthesis. Five exceptional mutants were partially defective for the synthesis of both and appear to be leaky. Complementation tests with tandem duplications suggest that the mutations causing the Cys auxotrophy affect only one cistron. The cysG gene is transcribed in a clockwise direction; this was demonstrated by a method that permits determining the orientation of two genes of unknown orientation provided their relative map order is known. The cysG gene was not part of the cysteine regulon, but had a substantial basal level of expression which was induced fivefold when cells were grown anaerobically on nitrite. Finally, we used Mud-generated duplications to genetically determine the organization of the cysG and nirB genes.
The biosynthesis of cysteine is central to the assimilation of sulfur in the facultative anaerobic bacteria Escherichia coli and Salmonella typhimurium (22). Assimilation occurs by the reduction of sulfate (SO42-) through sulfite (SO32-) to sulfide (S2-) (14, 15, 22); reduced sulfide reacts with O-acetyl-serine to form cysteine. All reduced sulfur in the cell is derived from cysteine (22). The aerobic reduction of sulfate to sulfide has been well characterized, and mutants defective in each step of the pathway have been isolated. Sulfate is taken into the cell by the sulfate permease which is encoded by the genes at the cysA locus (30). Internal sulfate is reduced to sulfite in three sequential steps and requires the products of the cysH, cysD, and cysC genes, which encode 3'-phosphoadenosine 5'-phosphosulfate reductase, ATP sulfurylase, and 5'-phosphosulfate kinase, respectively (14, 15). Sulfite is reduced in one step to sulfide; this step requires the cysi and cysI genes, which encode sulfite reductase (14, 15). The cysJ and cysI genes, as part of the cysteine regulon, are coordinately regulated in response to cysteine levels (22). In addition to the cysJ and cysI genes, the cysG gene is also required for sulfite reduction, this gene encodes a protein involved in the synthesis of siroheme, an Fe2+-containing cofactor of sulfite reductase which is also found in the NADH-dependent nitrite reductase of E. coli (10, 24, 25). Strains with a cysG mutation are cysteine auxotrophs and are unable to use nitrite (NO2-) as a nitrogen source during anaerobic growth; they are phenotypically Cys- Nir- (24). In S. typhimurium, a few cysG mutants have been tested for the ability to synthesize cobalamin (B12), and all show a Cob- phenotype (20). This is expected since cobalamin and siroheme are derived from a common precursor, dihydrosirohydrochlorin (DSC) (2). In E. coli, the cysG gene has *
been shown to encode an S-adenosylmethionine methyltransferase and to be in an operon with another gene(s) necessary for nitrite reductase activity (25, 31, 32, 39, 40). The proposed biosynthetic pathways of heme, siroheme, and B12 in S. typhimurium are shown in Fig. 1. The biosynthetic pathway for siroheme branches from that of heme at uroporphyrinogen III (URO III); this heme precursor is converted to DSC. In the formation of DSC, URO III is methylated at two sites (2). In siroheme synthesis, DSC is oxidized to yield sirohydrochlorin (SC). The oxidation step occurs spontaneously in vitro under aerobic conditions, but may require catalysis in vivo (2). In the third and final step, the Fe2" is chelated to form siroheme (28, 29, 35). This scheme predicts two classes of mutants able to make heme but unable to make siroheme. These mutants should all be detected as cysteine auxotrophs since siroheme is essential for sulfite reduction. The first class would be blocked in the formation of DSC and unable to synthesize either siroheme or B12. The other classes should be blocked after DSC; these should be defective in sulfite and nitrite reduction and retain the ability to synthesize B12. The only heme-positive siroheme-negative mutants isolated so far were detected as cysteine auxotrophs of the first class; their mutations (cysG) map at min 72 of the chromosome. Of the few cysG mutants previously tested, all were defective for both B12 and cysteine synthesis. Since cysG is the only locus for siroheme-defective mutants among the large number of cys auxotrophs studied in S. typhimuriuM (14, 15) and since only a few cysG mutants have previously been tested for B12 synthesis, it seemed possible that the whole pathway of siroheme synthesis might be encoded at the cysG locus. To check this possibility and to further investigate regulation of the cysG gene(s), we initiated a genetic investigation of this locus, which serves both B12 and siroheme synthesis. We isolated a set of mutations blocked for the synthesis of siroheme (Cys- Nir-) and determined their ability to syn-
Corresponding author.
t Present address: Section of Microbiology, Wing Hall, Cornell
University, Ithaca, NY 14853-8101.
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GOLDMAN AND ROTH
J. BACTERIOL. cool
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SIROHYDROCHLORIN FIG. 1. Synthesis of siroheme from aminolevulinic acid in E. coli and S. typhimurium. Aminolevulinic acid (ALA) is converted in two steps to URO III, the last intermediate common to both heme and siroheme synthesis. URO III is methylated at the C-2 and C-7 carbons to form DSC. DSC, the last common intermediate in both siroheme and B12 synthesis, is oxidized to form SC. An Fe2+ is then chelated to SC to form siroheme, the cofactor of both sulfite and nitrite reductase.
thesize B12. A deletion map of these mutations was constructed, and complementation tests were done. We found that all cysG mutants were defective for B12 synthesis and that only one complementation group necessary for cysteine synthesis exists at the cysG locus. We characterized the transcriptional regulation of this gene using Mud::lac fusions. We report here that the cysG gene was not regulated in response to cysteine levels and that its expression did not require the CysB activator protein. Therefore, the cysG gene is not part of the cysteine regulon. Transcription of the cysG gene was induced in response to nitrite when cells were grown under anaerobic conditions. Molecular analysis of the cysG gene in E. coli suggests that the gene is in an operon with the nirB gene, which encodes the structural gene for the NADH-dependent nitrite reductase (25, 31, 32). Sequence analysis of the cysG gene suggests that this is also the case in S. typhimurium (41). We used Mud-generated duplications to show genetically that the cysG and nirB genes are in an operon and that the cysG gene is at least partially expressed from the nirB promoter.
MATERIALS AND METHODS Bacterial strains and genetic methods. All strains used in this study are derived from S. typhimurium LT2 (Table 1). Two derivatives of bacteriophage Mu were used as transposons to form operon fusions; both are derived from the
phage MudI (Ampr lac cts) (6). The first derivative, MudIl-8, carries two mutations which make it conditionally defective for transposition (7). In this report, MudI1-8 (Ampr lac cts) will be referred to as MudA. The second derivative, MudI1734, specifies kanamycin resistance (Kanr) and lacks the Mu A and B genes necessary for transposition (18). In this report, MudI1734 (Kanr lac cts) will be referred to as MudJ. The TnJO and TnlOdTet insertion mutations were obtained from a pool of random insert mutants. Deletion mutations in cysG were isolated as tetracycline-sensitive derivatives of the TnlOdTet strain TT15028 and of the TnlO strains 171413 and FT1427; these were selected on BochnerMaloy medium (5, 26). The selection and screening of Tets derivatives was done at 42°C. Transductional crosses were performed as described previously (13). Duplications were generated by the method of Hughes and Roth (18). Media. Difco nutrient broth (NB, 0.8%) containing NaCl at a final concentration of 85 mM was used as the complex medium. The minimal medium was the E medium of Vogel and Bonner supplemented with 11 mM glucose, 22 mM glycerol, or 11 mM lactose (38). The sulfur-free minimal medium was NCE (38) supplemented with glucose or glycerol, 1.0 mM MgCl2, 40 mM citrate (trisodium salt), and the various sulfur sources as indicated. The nitrogen-free medium was NCN (38) supplemented with glucose, MgCl2, citrate, cystine, and either ethanolamine (0.2%; Aldrich) or 3 mM nitrite. Dimethylbenzimidazole (0.3 mM) was added to NCN medium when ethanolamine (EA) was used as the
S. TYPHIMURIUM cysG GENE
VOL. 175, 1993
1459
TABLE 1. Strain lista
TT1413 TT1427 TT9507 TT9521
1T9524 TT9641 TT9642 TT10607 TT10786 TT13506 TT13507 TT14746 TT14747 TT15028 TT15047 TT15048 TT15049 TT15052 TT15053 TT15126 TT15815 TT15816
cysJ1572::MudA
cysGl573::MudA fnr2::TnlO aroE568::MudA cysG3170::MudA cysG3168::MudA cysG3169::MudA cysG3174::MudA zhc-3665::TnlOdTet
AcysG3144/DUP861(aroE568)*MudA*(pyrE2681) DUP862(aroE568)*MudA*(pyrE2681) AcysG3172 lacZ cysG3168::MudA cysB3305::TnlOdTet
cysG3168::MudA metE205 ara-9 cysJ1572::MudA cysB3305::TnlOdTet nirPl::TnlO cysG::MudA nirB2::TnlO cysG::MudA DUP(aroE-cysG)*MudA*(aroE-cysG) fnr::TnlO cysG::MudA
Ff16899 TT16900 All strains
Laboratory collection C. Miller (37) Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection Laboratory collection This study This study This study This study This study This study Laboratory collection This study This study This study This study
Wild type fnr2::TnlOpepJ7::MudA leuBCD485 metE205 ara-9 cysGl5l8::TnlO cysGl526::TnlO pyrE2419::MudA pyrE2678::MudA pyrE2681::MudA
LT2 TN2063 TR6583
a
Reference or source
Genotype
Strain
are
derivatives of S. typhimurium LT2.
nitrogen source. Sulfur sources were added to the medium in the following concentrations: sulfate (1.0 mM), sulfite (0.3 mM), djenkolic acid (0.5 mM), and cystine (0.3 mM). Cyanocobalamin (Sigma Chemical Co.) was used as the exogenous B12 source (100 ,ug/liter). Auxotrophic supplements were added at final concentrations as described previously (13). Ampicillin was added to a final concentration of 30 ,ug/ml in NB and 15 ,ug/ml in NCE media. Tetracycline was added to a final concentration of 20 ,ug/ml in NB and 10 pg/ml in NCE media. Indicator plates containing X-Gal were made by adding 5-bromo4-chloro-3-indolyl-,-D-galactopyranoside to a final concentration of 25 ,ug/ml. Solid rich medium contained agar (1.5%; Difco) or, when minimal medium was used, Noble agar (1.7%; Difco). Cells were grown at 37°C. Scoring ability to reduce sulfite. The ability to reduce sulfite was scored as the ability to grow on minimal medium containing sulfite as the sole sulfur source. Alternatively, production of sulfide was scored directly by using bismuth sulfite plates (Difco). A black precipitate (Bi2S3) forms when bismuth interacts with the reduced sulfide. The latter is the more sensitive method for the detection of the ability to reduce sulfite. Chemical mutagenesis. Phage P22 lysates for localized mutagenesis were concentrated and mutagenized with hydroxylamine by the method of Hong and Ames (17). The survival rate of the phage particles from this mutagenesis was 0.1%. This mutagenized lysate, grown on strain Tf15028, was used to transduce LT2; among the Tetr transductants, cysG mutants were identified. Mapping of TnlO and TnlOdIet mutations. The TnlO mutations were mapped by the method of Benson and Goldman (3). Briefly, Tetr cells were streaked onto NB plates containing tetracycline. Single-colony isolates were
inoculated into liquid NB medium containing tetracycline and grown to the mid-exponential phase. Cells were diluted 1/10, and 0.1 ml was plated onto Bochner-Maloy medium (5, 26). Portions (5 ,ul) of MudP22-transducing lysates were spotted onto the plates which were then incubated overnight at 42°C. The TnJO mutations were located by determining which lysate produced the greatest growth on BochnerMaloy medium after 1 to 2 days of incubation. Once the general region of the chromosome was determined, transductional linkage to known markers was tested. Deletion mapping. The endpoints of deletions were determined by selecting for recombinants in crosses between two cysG auxotrophs. Fifteen microliters of concentrated lysate was mixed with 150 RI of cells. This mixture was allowed to incubate for 30 min at room temperature and was then plated onto minimal plates containing low amounts of cystine (6 ,uM). The Cys+ recombinants were scored after incubation for 48 h at 37°C.
fP-Galactosidase
assay.
1-Galactosidase activity
was as-
sayed as described by Miller (27), using CHCl3-sodium dodecyl sulfate to permeabilize whole cells. Enzyme activity is expressed as nanomoles of o-nitrophenyl-o-D-galactoside per minute per unit of optical density at 650 nm (1 unit = 1 ml of cell culture with an optical density at 650 nm of 1). All assays were performed in duplicate with early to midexponential-phase cultures. Growth under anaerobic conditions. Cells were grown anaerobically on solid medium in an anaerobic chamber (model 1024; Forma Scientific) whose atmosphere contained nitrogen-hydrogen-carbon dioxide in a percentage of 90-5-5. Oxygen levels were determined with an oxygen detector (model 10 gas analyzer; Coy Laboratory Products). For anaerobic growth in liquid medium, oxygen was removed by the method of Balch and Wolfe (1). Carbon, nitrogen, and
1460
GOLDMAN AND ROTH
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FIG. 2. Deletion map of the cysG gene. A fine-structure deletion map of the cysG gene was generated to assist in the analysis of the locus, as described in Materials and Methods. All numbers listed on the map are cysteine auxotrophic alleles. Open triangles represent TnlO insertion mutations, and closed triangles represent MudA insertion mutations. Arrows represent the orientation of the MudA insertion mutations. Open squares represent leaky alleles. The origin of replication is to the right on this map, and the gene is transcribed from left to right.
sulfur sources were added to sterile medium with a syringe. Such tubes were inoculated by injecting 0.1 ml of an overnight culture grown aerobically to saturation in NCE medium containing glucose, citrate, MgCl2, and cystine. RESULTS Isolation and deletion mapping of cysG deletion and point mutations. The proposed biosynthetic pathway for cobalamin (B12) and siroheme suggests that there should be two classes of siroheme auxotrophs (Fig. 1). The first class would be blocked in the formation of DSC, a precursor of B12, and be defective for cysteine and cobalamin biosynthesis and nitrite reduction (Cys- Nir- Cob-). The second class would be blocked after synthesis of DSC; they should be CysNir- but retain the ability to synthesize B12. Since all previously isolated Cys- Cob- mutants were detected as cysteine auxotrophs and map at min 72, it seemed possible that the whole pathway of siroheme synthesis might be encoded by the cysG locus. Only a few cysG mutants had previously been isolated and even fewer tested for B12 synthesis. We isolated a set of cysG point and deletion mutants as described in Materials and Methods and characterized their phenotypes with respect to their ability to make B12 (Cob) and use nitrite (Nir). A deletion map of the cysG locus was generated, as described in Materials and Methods, and the phenotype conferred by each deletion and point mutation was correlated with its map position (Fig. 2). We also analyzed 14 insertion mutations with respect to phenotype and map position. Phenotypes of cysG mutants. The ability of cysG mutants to reduce sulfur was tested as described in Materials and Methods. Of the 66 cysG mutants tested, none of the 34 deletion and insertion mutants were able to reduce sulfite. Five point mutants were unable to grow on sulfite as a sole sulfur source but were positive in the bismuth assay for sulfite reduction. They appear to be only partially defective for sulfite reduction. The ability of cysG mutants to reduce nitrite was scored as the ability of cells to grow anaerobically on solid medium containing nitrite as the sole nitrogen source as described in Materials and Methods. Of the 66 cysG mutants tested, only those five mutants with a partial defect in sulfite reduction were able to use nitrite as a
nitrogen source. The rest were unable to use nitrite but grew well when ammonia was added to the medium. To determine the ability of cysG mutants to synthesize B12, we introduced a metE mutation into each cysG auxotroph. The metE gene encodes homocysteine methyltransferase, the last enzyme in the methionine biosynthetic pathway (23). The auxotrophy of metE mutants is corrected in the presence of cyanocobalamin, because cobalamin permits the activity of an alternative B12-dependent transferase, encoded by the metH gene (12, 23). Under anaerobic conditions, Cob' Salmonella strains produce B12, activate the metH gene, and synthesize methionine despite the metE mutation (16, 20). The ability of cysG metE strains to synthesize B12 was scored as their ability to grow without methionine under anaerobic conditions. The metE mutant grew anaerobically without methionine, while the cysG metE double mutants could not grow anaerobically unless the medium was supplemented with B12. This showed that the cysG mutant was blocked for B12 synthesis. All cysG deletion and insertion mutants and most point mutants proved to be unable to synthesize B12. The same five mutants (described above) that showed a leaky Cysphenotype appeared to synthesize B12 by virtue of residual CysG activity because they grew anaerobically without methionine. A second, more stringent, test for the ability to make B12 was anaerobic growth on ethanolamine (EA), which can be used anaerobically as a nitrogen source if B12 is present to permit activity of EA ammonia lyase. All 66 cysG mutants were tested for the ability to use EA as a nitrogen source anaerobically. Most cysG mutants did not grow unless provided with B12; only the five leaky cys auxotrophs described above were able to use EA as a nitrogen source. We did not recover cysG mutants defective in conversion of DSC to siroheme (Cys- Nir- Cob'). It seemed possible that the cysG region might include a set of genes involved in the last steps of siroheme synthesis, if such genes were located promoter-proximal to the methylase gene. If this were the case, it might be possible to isolate revertants that remove the polar block, generating strains with a Cob' siroheme- phenotype by relieving polarity. Several attempts to isolate such mutants were unsuccessful.
S. TYPHIMURIUM cysG GENE
VOL. 175, 1993
cysG::Mu dA (minute 72)
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FIG. 3. Transcriptional orientation of the cysG and pyrE genes. The direction of transcription of both the cysG and pyrE genes is determined by using the mixed lysate assay to generate duplications with strain LT2 as the recipient. The Lac phenotype of the resulting duplication-containing recombinant is then scored. (A) The chromosome if the cysG and pyrE genes are transcribed convergently; (B) the chromosome if the genes are transcribed divergently. Arrows represent the proposed orientation of transcription. The numbers and letters by the genes are arbitrary designations to clarify gene orientation. The top half of the figure shows how the transducing fragment is made, and the bottom half of the figure shows how the transducing fragment recombines with the recipient chromosome at a replication fork. The duplications strains were all Lac-, and therefore the genes are transcribed convergently.
Orientation of cysG gene transcription. The orientation of the cysG gene was determined by the method of Hughes and Roth (18), by generating duplications using mixed lysates of strains carrying MudA insertions, as described in Materials and Methods. Mud-generated duplications form by recombination between insertions in the same orientation. We found that duplications formed when a lysate made on Lac' cysG::MudA strain TT13507 was mixed with a lysate made on the Lac- pyrE::MudA strain TT9507 and used to transduce LT2 to Ampr. Duplications were also generated when Lac- cysG::MudA strain T714746 and Lac' pyrE::MudA strain 1T9521 or TT9524 were used as donors in the same cross. Mud-generated duplications never occurred when Lac' cysG::MudA strain T713507 and Lac' pyrE::MudA strain TT9521 or TT9524 were used as donors or when Lac- cysG::MudA strain TT14746 and LacpyrE::MudA strain 119507 were used as donors in this cross. Five cysG::MudA (four Lac' and one Lac-) fusions and three pyrE::MudA (one Lac' and two Lac-) fusions were tested in this manner. All four of the Lac' cysG::MudA fusions were oriented in the same direction; all are in opposite orientation to the Lac- cysG::MudA fusion
(data not shown). The cysG andpyrE genes are thus oriented in opposite directions. This test by itself gives no information whether that direction is convergent or divergent relative to the chromosome. Although the orientation of thepyrE gene is unknown, the direction of transcription of these genes relative to each other, and thus their orientation in the chromosome, can be determined from the Lac phenotype of the Mud: :lac element at the join point of the duplications generated above. This determination depends on knowing the map positions of the two loci; the cysG gene maps at 72 min and the pyrE gene maps at 79 min (34). If the genes are transcribed toward each other, as diagrammed in Fig. 3A, the duplication strains will always be Lac- and have little or no P-galactosidase activity because no promoter is present at either side of the join point to express the lac genes. If the genes are transcribed away from each other (Fig. 3B), the final duplication strain will be Lac' because a promoter faces both ends of the Mud::lac element at the join point. Note in Fig. 3 that, although the fusions have been drawn in one direction, the orientation of the particular lacZ gene fusions used to generate the duplication is irrelevant to the Lac phenotype of the duplication
1462
GOLDMAN AND ROTHJ.ATEOL J. BACTERIOL.
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I
Recipient duplication recombinant Aro+, Cys-, Pyr+, Amp FIG. 4. Generation of heteroallelic strain for complementation analysis. A strain containing a small deletion at the promoter-distal end of the cysG gene is used as a recipient to generate a strain containing a duplication of the cysG gene. The resulting strain (TT15047) is used as a recipient in a cross with a strain containing a Tn1OdTet insertion 90% linked to a cysG point mutation as described in Materials and Methods. Heteroallefic recombinants are scored for the ability to use sulfite as the sole sulfur source. strain. In our experiments, the duplication strains formed phenotypically Lac- and expressed less than 2 U of 13-galactosidase activity regardless of which donors were used (data not shown). The genes were thus transcribed convergently. That is, cysG transcription is clockwise and pyrE transcription is counterclockwise. In previous studies, the direction of transcription of a gene or operon using mixed lysates has been determined by comparing the orientation of an unknown gene with that of a known gene (18). Analyzing the lactose phenotype of the duplication strain allows one to determine the direction of transcription of two unknown genes if the relative map positions of the two genes are known. Orienting the cysG genetic map Vis-a-vis the direction of transcription. The direction of transcription relative to the cysG genetic map was determined in two ways. In the first, three cysG::TnlO cysG::MudA double mutants were constructed to test whether these TnlO insertions were polar on lacZ expression from the cysG::MudA insertion. Mutants containing the cysG3l7O::MudA (TT13506) allele alone and all the cysG::TnlO cysG::MudA double mutants were phenotypically Lac', suggesting that the Mud elements are upstream of the TnlO insertions. Furthermore, the P-galactosidase activity of the double mutants was the same as that of the MudA insertion alone (data not shown). Apparently all the TnlO insertions tested are promoter distal from the MudA insertion. This suggests that the gene is transcribed as the map is drawn in Fig. 2. In the second test, strain TT14746, a Lac- cysG::MudA insertion, was used to select for Lac' revertants. It was presumed that a Lac- insertion was oriented such that transcription from the cysG promoter would cross the lac operon in the wrong orientation. Some Lac' revertants could arise by means of deletions, which fuse a foreign promoter to the lacZ gene. Lac' revertants of TT14746 were found at a frequency of 1/108. Eleven independent Lac' revertants were isolated in this manner. All were tested, by recombinational analysis, for loss of cysG material. Two of the Lac' revertants carried deletions of all the material to the right of the cysG: :MudA insertion. One of these is listed in the deletion map as cysG3J172. None of the Lac' revertants carried detectable deletions to the left of the cysG::MudA insertion, as determined by recombinational analysis. Thus, the direction in which the deletions were generated and the lack of polarity of Tn1O insertions on the cysG: :MudA insertion strongly suggest that the gene is transcribed as depicted in Fig. 2. Complementation tests of cysG mutants. The above results suggest that all cysG mutants are blocked for the synthesis of were
DSC; none appeared to have a defect limited to either of the last two steps of siroheme synthesis. It could be argued that siroheme genes are all located at the cysG locus but are essential. We believe this is unlikely since deletions that remove the entire region could be generated. It was also possible that the cysG locus encodes all siroheme genes but that all mutations in the later steps also lack the first activity. Alternatively, siroheme might be an essential cofactor required for later steps in the synthesis of cobalamin. To explore the possibility of multiple genes at the cysG locus, we did complementation tests. Complementation tests were done by using a tandem duplication of the chromosomal segment between the pyrE and arcE loci by the method of Hughes and Roth (18). Heteroallelic cysG mutants were generated by introducing the pyrE-aroE duplication into deletion mutant cysG3J44. The cysG deletion affects the promoter-distal end of the genetic map of the cysG locus (Fig. 2) and should therefore not show a polar effect on the rest of the cysG region. The duplication formed carries the cysG deletion in both copies of the duplicated region. The diploid Cys- Ampr strain was transduced to Tetr with phage grown on strains carrying a Tnl0dTet insertion (zhc3665) 90% linked to the cysG gene and containing one of the point mutations listed on the cysG map (Fig. 2). Figure 4 shows the three possible outcomes of the cross. Class one transductants inherited only the TnlO element without the cysG point mutation and remained Cys-. Class two transductants have a wild-type cysG gene. The third class inherited the donor point mutation in place of one copy of the deletion and became heterozygous for the cysG locus; 90% of the Tetr transductants were of the third class and became heterozygous for the cysG mutations. Of 27 tight point mutations tested, none showed complementation with the small cysG deletion used in these tests. We conclude that the mutations causing cys auxotrophy affect only one gene. The cysG gene is not in the cysteine regulon. Since siroheme is required for cysteine synthesis, one might expect that the cysG gene would be coregulated with the cysteine biosynthetic genes. The cysi and cysI genes, which encode sulfite reductase, are part of the cysteine regulon. Expression of these genes requires a positive regulatory protein, encoded by the cysB gene, which is activated by sulfur limitation (21, 22). Growth of a cysteine auxotroph on djenkolic acid, a poor source of reduced sulfur, causes derepression of the genes of the cysteine regulon (22). A 1'(cysG-lacZ) fusion strain (T1113506) was used to study the transcriptional regulation of the cysG gene (Table 2). Expression of the cysG gene was not derepressed when a
VOL. 175, 1993
S. TYPHIMURIUM cysG GENE
TABLE 4. Expression of CF(cysG-lacZ) fusion strain in various backgroundsa
TABLE 2. 3-Galactosidase levels of 4'(cysG-lacZ) and 4d(cysJ-lacZ) fusions under indicated growth conditionsa 13-Galactosidase leveP Fusion
Genotype
Aerobic Anaerobic DjenDjen~~Djenkolic Cystine kolic Cystine acid acid
40(cysG-lacZ)
cysB+
1(cysJ-lacZ)
cysB::TnlOdJret 40 cysB+ 1,360 cysB::TnlOdTet
